The major goal of this proposal is to better understand how Ras proteins couple extracellular signals to intracellular signaling pathways. Ras proteins were first identified by their ability to act as oncogenes and influence cell proliferation. More recently, it has become clear that Ras proteins contribute to signaling pathways that regulate many other cellular processes, including differentiation, and functions displayed by non-dividing fully differentiated cells. Most research on the regulation of Ras activity has focused on Ras activation by SOS nucleotide exchange factors. These proteins respond to surface tyrosine kinase stimulation by enhancing the GTP-bound state of Ras. Recently, we have discovered an alternative pathway to Ras that involves calcium/calmodulin-induced activation of Ras-GRF exchange factors. This pathway appears to be used extensively in neurons, but not exclusively there, because Ras-GRFs have been found in some other tissues. One aim of this proposal is to investigate the role of a newly identified Ras- GRF1 binding protein in targeting Ras-GRF1 to specific cellular sites. Since changes in calcium levels are known to be very localized in cells, correct targeting of Ras-GRF1 may be critical for it to respond to the appropriate calcium signal. The second aim is to identify which calcium signals activate Ras-GRF1, by studying the consequence of suppressing Ras-GRF1 activity in neurons. We have already produced a Ras-GRF1 knockout mouse for analysis. Other approaches will be to introduce dominant negative mutants of Ras-GRFs and antisense oligonucleotides into neuronal cultures. Another aim of this proposal is to identify cell components that function along with calmodulin to promote Ras-GRF activation by 1) their ability to reconstitute Ras-GRF activation in vitro and 2) their ability to bind to critical regions of Ras-GRF. Finally, experiments will be performed to determine if at least some of the differences in signaling initiated by surface tyrosine kinases and different types of calcium channels could stem from differential activation of individual Ras effector proteins. This goal may begin to reveal how Ras proteins can produce multiple effects in cells in a specific manner.